Surgical patients with critical disorders such as thermal injury, sever trauma, shock, and sepsis are commonly complicated with the gastrointestinal mucosal injury, which can lead to the development of systemic infection, septic shock, and multiple organ failure. Restitution is an important process for repair of disrupted intestinal lining. Previous studies have suggested that intestinal epithelial restitution is delayed in critically ill individuals. Our recent work has revealed that milk fat globule-EGF factor 8 (MFG-E8) is a macrophage-derived intestinal epithelial restitution factor. MFG-E8 promotes intestinal epithelial wound-healing via a PKC5 dependent manner. In sepsis, intestinal injury is associated with downregulation of MFG-E8, which contributes to the impairment of restitution. In this proposal, therefore, we will test a central hypothesis that under the physiological state, MFG-E8 maintains intestinal epithelial cell migration via a typical signal pathway, whereas under the septic condition, inflammatory mediators down-regulate MFG-E8 expression, which in turn impairs intestinal epithelial cell restitution. In Specific Aim 1, we will define the molecular and signaling mechanism through which MFG-E8 enhances intestinal epithelial restitution. Our preliminary data suggest that MFG-E8 activates several PKC5-associated intracellular signal molecules. Therefore, we will investigate the role of these molecules in MFG-E8-induced intestinal epithelial cell migration using molecular biology and gene knockdown approaches. In Specific Aim 2, we will focus on understanding the cause of down-regulation of intestinal MFG-E8 gene expression and impairment of MFG-E8-dependent intestinal restitution in sepsis. Our preliminary data suggest that a sepsis-derived inflammatory mediator mimics septic effect on intestinal MFG-E8 gene expression and restitution. Thus, we will test the hypothesis that this mediator inhibits intestinal MFG-E8 gene expression, which subsequently results in impairment of intestinal restitution. In Specific Aim 3, we will investigate the molecular mechanism underlying the inhibition of MFG-E8 expression in macrophages by septic insult-derived inflammatory stimulus. Our preliminary data show that repression of MFG-E8 gene expression by an inflammatory stimulus is involved in affecting the activities of promoter and mRNA 3'-untranslated region (3'-UTR) of MFG-E8. To follow up on these findings, we will characterize binding motifs in the MFG-E8 gene promoter and their binding proteins, and study their role in suppression of MFG-E8 gene expression during inflammation. The standard approach for analysis of the gene promoter function will be applied. In addition, we will use the microRNA and site-directed mutagenesis technologies to study the role of 3'-UTR and microRNAs in regulation of MFG-E8 gene expression by an inflammatory stimulus. Achievement of these specific aims and confirmation of our hypotheses will provide the novel mechanistic information that allows us to understand the crucial role of MFG-E8 in the gut and its physio/pathological implications. Our studies will ultimately lead to development of novel strategies for management and treatment of intestinal injury in critically ill patients.